Hydrogel formulations

ABSTRACT

A polymeric prodrug composition including a hydrogel, a biologically active moiety and a reversible prodrug linker. The prodrug linker covalently links the hydrogel and the biologically active moiety at a position and the hydrogel has a plurality of pores with openings on its surface. The diameter of the pores is larger than that of the biologically active moiety at least at all points of the pore between at least one of the openings and the position of the biologically active moiety.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.10/960,851 filed on Oct. 7, 2004, and published as U.S. Publication No.2006-0002890 A1, which further claims priority under the provisions of35 USC §119 of United Kingdom Patent Application No. 0415041.3 filedJul. 5, 2004 and European Patent Application No. 04019303.9 filed Aug.13, 2004. The disclosure of each application is hereby incorporatedherein in its respective entirety, for all purposes.

FIELD

The present invention is directed to hydrogel depot formulations ofbiologically active moieties. The hydrogel depot formulations comprisebiologically active moieties such as peptides, proteins,oligonucleotides or polynucleotides, natural products or syntheticchemical compounds linked reversibly to mesoporous hydrogels.

BACKGROUND OF THE INVENTION Definitions

Micropore:

Pore in a three-dimensional polymer network that is smaller than a givenbiologically active moiety (smaller than 1 nanometer (nm))

Mesopore:

Pore in a three-dimensional polymer network that is larger than a givenbiologically active moiety (dependent upon the size of the biologicallyactive moiety, but usually larger than 1 nm, and smaller than 100 nm)

Macromonomer:

A polymer or oligomer whose molecules each have at least onepolymerizable functional group, often at the end or at the ends, thatenables it to act as a monomer. After polymerization, the groups arepart of the main chain of the final polymer.

Homopolymerization or copolymerization of a macromonomer yields comb,graft, or cross-linked polymers.

Crosslinking:

A reaction involving pairs of polymer chains that results in theformation of regions in a polymer from which at least four chainsemanate.

The region may be an atom, a group of atoms, or a number of branchpoints connected by bonds, groups of atoms, oligomeric, or polymericchains.

Biodegradable Polymer:

A polymer susceptible to degradation under in vivo conditions. In vivoconditions include, but are not limited to, degradation by enzymatic orchemical means under conditions present in a living body. Degradation isdefined as a chemical change in a polymeric material, accompanied bycleavage of chemical bonds in the polymer and a lowering of its molarmass.

Reactive Polymer:

A polymer having reactive functional groups that can be transformedunder the conditions required for a given reaction or application.

Hydrogel:

A hydrogel may be defined as a three-dimensional, hydrophilic oramphiphilic polymeric network capable of taking up large quantities ofwater. The networks are composed of homopolymers or copolymers, areinsoluble due to the presence of covalent chemical or physical (ionic,hydrophobic interactions, entanglements) crosslinks. The crosslinksprovide the network structure and physical integrity. Hydrogels exhibita thermodynamic compatibility with water that allows them to swell inaqueous media. The chains of the network are connected in such a fashionthat pores exist and that a substantial fraction of these pores are ofdimensions between 1 nm and 1000 nm.

Prodrug:

A prodrug is any compound that undergoes biotransformation beforeexhibiting its pharmacological effects. Prodrugs can thus be viewed asbiologically active moieties (such as drugs) containing specializednon-toxic protective groups used in a transient manner to alter or toeliminate undesirable properties in the parent molecule.

Carrier-Linked Prodrug (Carrier Prodrug):

A carrier-linked prodrug is a prodrug that contains a temporary linkageof a given active substance with a transient carrier group that producesimproved physicochemical or pharmacokinetic properties and that can beeasily removed in vivo, usually by a hydrolytic cleavage.

Cascade Prodrug:

A cascade prodrug is a prodrug for which the cleavage of the carriergroup becomes effective only after unmasking an activating group.

Protein Depots

Among the first polymers employed for protein drug delivery applicationswere polylactide-coglycolides (PLGA). These materials are ratherhydrophobic, and only few protein and peptide drugs could be formulatedinto delivery systems (e.g., somatropin, Nutropin Depot; triptorelin,Trelstar™ Depot; octreotide, Sandostatin® LAR®; leuprolide, LupronDepot®). The hydrophobic nature of PLGA is exploited in the productionprocess of such PLGA-protein formulations. PLGA is provided as asolution in a water-miscible organic solvent, protein is dissolved inwater, and the two solutions are mixed in a mixing step. As aconsequence, PLG precipitates and physically entraps the protein in aprecipitate. The precipitate has a low water content, and pore sizes aresmaller than 1 nm and do not exhibit hydrogel-like properties. Dependenton the conditions of the mixing step, the water-miscible organic solventused and the physicochemical properties of the protein, the loading ofthe precipitate of the PLGA and the protein may vary greatly.

Furthermore, Dong Hee Na, et al. showed that upon degradation of theprecipitates the encapsulated protein and peptide drugs are chemicallymodified by acylation resulting in the release of modified drug moieties(see Dong Hee Na, et al., 2003, J. Contr. Release 92, 291-299).

In order to address a fundamental shortcoming of PLGA-precipitates,recent developments focused on the use of hydrogels for proteindelivery. Hydrogels are promising materials for drug deliveryapplications, in particular for the delivery of peptide, protein,oligonucleotide or polynucleotide drugs (“biotherapeutics”). Thesebiotherapeutics are fragile macromolecules which often require awell-hydrated environment for activity and structural integrity. Thehigh water content of the hydrogels renders the material biocompatibleand minimizes inflammation reactions of tissue in contact with thehydrogel. Especially for the delivery of protein therapeutics, the highdegree of hydration may help to preserve the folding of the proteinwhich is a prerequisite for its bioactivity. In hydrophobicenvironments, proteins tend to denature and aggregate and lose activity.

Two different approaches for the preparation of hydrogel-based depotsare known in the art, non-covalent depots and covalent depots.

In the non-covalent approach, biologically active moieties such as drugsare encapsulated physically without chemical linkage to the hydrogel.For this approach, the average pore size in the three-dimensionalnetwork of the hydrogel has to be smaller than the size of thebiologically active moiety for efficient encapsulation by the hydrogel.Therefore, the biologically active moiety can not be incorporated intothe hydrogel after hydrogel formation. In the non-covalent approach, thehydrogels have to be chemically crosslinked in the presence of thebiologically active moiety or pores have to be formed through physicalcrosslinks in a self-assembly process, also in the presence of thebiologically active moiety. The size of the pore size is the key factorgoverning the encapsulation of the biologically active moiety. If thepores are larger than the biologically active moiety, then thebiologically active moiety will rapidly effuse out of the interior ofthe hydrogel (so-called “burst” release). Therefore the crosslinking isallowed to proceed to such extent that a hydrogel with pores is formed,and the biologically active moiety is physically entrapped inside thepores.

The size of the pore in a chemically crosslinked hydrogel may bedetermined by the measurement of the diffusion of different moleculeswith known sizes (for example a set of different proteins) into thehydrogels. For example, this can be done experimentally by sizeexclusion chromatography in which the hydrogel is shaped in bead formand packed into a size exclusion chromatography column. Once thehydrodynamic diameter of the protein is larger than the pores in thehydrogel, no diffusion of the protein into the beads made of thehydrogel can take place and the protein elutes in the exclusion volumeof the exclusion chromatography column.

The size of the pores in self-assembled networks is difficult to measuredue to the structural instability of self-assembled networks, which isdue to the usually weak physical interactions within the self-assemblednetwork.

The hydrogels can be prepared by crosslinking hydrophilic biopolymers orsynthetic polymers. Examples of the hydrogels formed from physical orchemical crosslinking of hydrophilic biopolymers, include but are notlimited to, hyaluronans, chitosans, alginates, collagen, dextran,pectin, carrageenan, polylysine, gelatin or agarose. (see.: W. E.Hennink and C. F. van Nostrum, Adv. Drug Del. Rev. 2002, 54, 13-36 andA. S. Hoffman, Adv. Drug Del. Rev. 2002, 43, 3-12). These materialsconsist of high-molecular weight backbone chains made of linear orbranched polysaccharides or polypeptides.

Examples for drug-biopolymer hydrogel encapsulation include theencapsulation of recombinant human interleukin-2 from in chemicallycrosslinked dextran-based hydrogels (J. A. Cadee et al., J Control.Release. 2002, 78, 1-13) and the encapsulation of insulin in anionically crosslinked chitosan/hyaluronan complex (S. Surini et al., J.Control. Release 2003, 90, 291-301)

Examples of hydrogels based on chemical or physical crosslinkingsynthetic polymers include but are not limited to(meth)acrylate-oligolactide-PEO-oligolactide-(meth)acrylate,poly(ethylene glycol) (PEO), poly(propylene glycol) (PPO), PEO-PPO-PEOcopolymers (Pluronics), poly(phosphazene), poly(methacrylates),poly(N-vinylpyrrolidone), PL(G)A-PEO-PL(G)A copolymers, poly(ethyleneimine), etc. (see A. S Hoffman, Adv. Drug Del. Rev 2002, 43, 3-12).

Examples for protein-polymer encapsulation include the encapsulation ofinsulin in physically crosslinked PEG-g-PLGA and PLGA-g-PEG copolymers(see B. Jeong et al. Biomacromolecules 2002, 3, 865-868) and theencapsulation of bovine serum albumin in chemically crosslinkedacrylate-PGA-PEO-PGA-acrylate macromonomers (see A. S. Sawhney et al.,Macromolecules, 1993, 26, 581-587)

This first non-covalent approach has several drawbacks. As thepolymerization or crosslinking step to form the hydrogel has to becarried out in the presence of the biologically active moiety (i.e. aprotein), the biologically active moiety is exposed to solvents andpolymerization reaction conditions which may cause denaturation orchemical modification of the biologically active moiety. Furthermore,the quality of the end product is difficult to control and batch tobatch variations may occur. Additionally, the loading of the hydrogelswith the biologically active moiety is usually rather low (<15% protein)and is difficult to control.

A further drawback of the non-covalent type of the hydrogels is theso-called burst effect. The burst effect is characterized by a fast anduncontrolled initial release of a weakly-bound biologically activemoiety from the hydrogel. The initial burst release can account for upto 20% of the encapsulated biologically active moiety.

As in PLGA precipitates, degradation of the hydrogels is required forrelease of the biologically active moiety from the crosslinkedhydrogels. Self-assembled systems may also depend on degradation ordisaggregation for release of the biologically active moiety.Degradation of the hydrogel increases the size of the pore to the extentthat the biologically active moiety may diffuse out of the interior ofthe hydrogel into surrounding body fluids to exert its bioactivity.Degradation of the hydrogel is a process which is dependent on a numberof parameters, some of which are not well understood. As the degradationof the hydrogel is dependent upon in vivo conditions, there may besignificant contribution of complex biodegradation processes to theoverall degradation of the hydrogel.

The small size of the pore may reduce the water content of the hydrogeland therefore its compatibility with fragile biomolecules.

It is difficult to optimize release kinetics in vivo, which depend inturn on the conditions of the copolymerization process carried out inpresence of the biologically active moiety. This poses altogether asignificant obstacle for the successful development of these types ofdrug delivery systems.

Other inherent drawbacks of degradation-dependent release of thebiologically active moiety are interpatient and injection sitevariability when the degradation is catalyzed by enzymes. Enzyme levelsand specificities vary greatly between patients and also in dependenceon the tissue chosen for injection and other difficult-to-controlparameters such as the depth of needle insertion. Furthermore, lack ofcontrol over degradation typically may lead to burst effects.

Another complication lies in the fact that polymer degradation under invivo conditions may also occur chemically, without the contribution ofbiological factors such as enzymes. For instance, ester bonds typicallyemployed as biodegradable bonds (cleavable by esterases but also certainproteases) may spontaneously hydrolyze at the biological pH of 7.4 inplain buffer in the absence of ester-cleaving proteins. Typically,microporous hydrogels require a high amount of ester bonds in order toeffect efficient release of the biologically active moiety. Both thehigh local concentration of the ester bonds and the tight encapsulationof the biologically active moiety may lead to side reactions. It may bepossible that an amino group present in the biologically active moietymay be positioned in proximity to an ester group, with the amino groupproviding a nucleophile effecting ester cleavage and subsequentamidation. This process results in a very stable amide linkage betweenthe biologically active moiety and the polymer. The biologically activemoiety will not be released until the polymer chain to which thebiologically active moiety is attached, is degraded, and thebiologically active moiety will be permanently modified. Suchmodifications are known to reduce bioactivity of the biologically activemoiety and may also cause side effects, such as immunogenicity orcarcinogenicity. In addition, this undesirable modification process islargely uncontrolled and gives rise to a variety of molecular species.This type of side-reaction is described in Dong Hee Na et al., 2003, J.Contr. Release 92, 291-299.

In the alternative covalent depot approach, the biologically activemoiety (such as a drug molecule) is reversibly attached to the hydrogelby a covalent or ionic linkage. In this case a hydrogel with mesopores(a so-called mesoporic hydrogel) can be used. The release of thebiologically active moiety from the mesopores in the hydrogels isprevented by the attachment of the biologically active moiety.

There are only few examples describing this second approach ofreversibly linking the biologically active moiety to the hydrogel.

J. Harris and X. Zhao (in European Patent No. EP 1 053 019 B1) describethe reversible covalent attachment of a lysozyme protein to a hydrogelprepared by radical copolymerization of a PEO-diacrylate and alysozyme-modified PEO-monoacrylate. The lysozyme was coupled to thePEO-monoacrylate via a thiourea group and a short biodegradable esterlinker Release of the lysozyme from the hydrogel was effected byincubation in pH 7 buffer.

Upon cleavage of the ester bond in the approach described in the '019patent application, modified protein moieties are released as thecleaved linker moiety is still attached to the protein via the stablethiourea group. Furthermore, as the reaction of the activatedlinker-PEO-monoacrylate with the amino groups of the lysozyme is notregioselective, a variety of differently modified regioisomers arereleased, which is undesirable. Furthermore, as the encapsulation of theprotein is upon hydrogel formation by radical polymerization, theprotein can, in addition to the covalent attachment, also beencapsulated in pores of the formed three-dimensional network that aresmaller than the diameter of the protein. Therefore, the release of theprotein is not solely governed by cleavage of the linker but can also beinfluenced by the structure of the hydrogel.

Hubbell and coworkers (U.S. Patent Application Publication No.2003/0220245) described in a similar approach the reversible attachmentof a small synthetic peptide via a cysteine residue to anon-biodegradable hydrogel. The peptide was coupled to PEO-diacrylate bya Michael addition reaction. The peptide modified PEO-monacrylate wasradically crosslinked with a PEG-diacrylamide to form a peptide modifiedhydrogel. Release of the propionyl-modified peptide was effected byincubation of the hydrogel in pH 7.4 buffer at 37° C.

Due to the current use of microporous hydrogel materials in bothnon-covalent and covalent drug depots, numerous problems have hinderedthe development of a robust and reliable system for sustained drugdelivery from a depot formed from a hydrogel.

DETAILED DESCRIPTION OF THE INVENTION

It has now been surprisingly discovered that mesoporous hydrogels can beused as polymer carriers for drug depots if provided as a carrier in aprodrug system.

The invention is directed towards mesoporous hydrogel prodrugs (MHP) ofbiologically active moieties, such as drug molecules. The MHP may beadministered to a patient to form a depot inside the patient whichprovides for a sustained release of the biologically active moiety overa desired period of time.

A main advantage of the MHPs is to provide depot formulations withoutthe need for encapsulation. Until now, many biocompatible hydrogelmaterials with large pore sizes could not be used for drug formulationdue to their lack of encapsulating properties. In such biocompatiblehydrogel materials, biologically active moiety would be released toofast for most therapeutic applications from such well-hydrated andmechanically soft materials. The provision of a hydrogel as a prodrugcarrier according to the invention allows the development of superiordrug delivery systems. Carrier material properties such asbiocompatibility (minimal irritation, immunogenicity, toxicity) may beoptimized independently from the release properties of the biologicallyactive moiety as the release properties are solely governed by theprodrug linker cleavage kinetics. The release of the biologically activemoiety is therefore largely independent from the carrier material (i.e.,the hydrogel) and does not require chemical or enzymatic degradation ofthe hydrogel.

The MHP system consists of three parts, a mesoporous hydrogel carrier, aprodrug linker and a biologically active moiety, such as a drugmolecule. The prodrug linker is covalently bonded to the mesoporoushydrogel carrier and to the biologically active moiety in such a fashionthat the biologically active moiety-linker-hydrogel conjugate is acarrier prodrug.

Biologically Active Moiety

Suitable biologically active moieties include, but are not limited to,small organic molecule bioactive agents, biopolymers like proteins,polypeptides and oligo- or poly-nucleotides (RNA, DNA), and peptidenucleic acids (PNA).

Suitable organic small molecule bioactive drugs include, withoutlimitation, moieties such as central nervous system-active agents,anti-infective agents, anti-neoplastic agents, antibacterial agents,anti-fungal agents, analgesic agents, contraceptive agents,anti-inflammatory agents, steroidal agents, vasodilating agents,vasoconstricting agents, and cardiovascular agents. Non-exclusiveexamples of such compounds are daunorubicin, doxorubicin, idarubicin,mitoxantron, aminoglutethimide, amantadine, diaphenylsulfon, ethambutol,sulfadiazin, sulfamerazin, sulfamethoxazol, sulfalen, clinafloxacin,paclitaxel, moxifloxacin, ciprofloxaxin, enoxacin, norfloxacin, neomycinB, sprectinomycin, kanamycin A, meropenem, dopamin, dobutamin,lisinopril, serotonin, carbutamid, acivicin, etc.

Suitable proteins and polypeptides include, but are not limited to,ACTH, adenosine deaminase, agalsidase, albumin, alfa-1 antitrypsin(AAT), alfa-1 proteinase inhibitor (API), alteplase, anistreplase,ancrod serine protease, antibodies (monoclonal or polyclonal, andfragments or fusions), antithrombin III, antitrypsins, aprotinin,asparaginases, biphalin, bone-morphogenic proteins, calcitonin (salmon),collagenase, DNase, endorphins, enfuvirtide, enkephalins,erythropoietins, factor VIIa, factor VIII, factor VIIIa, factor IX,fibrinolysin, fusion proteins, follicle-stimulating hormones,granulocyte colony stimulating factor (G-CSF), galactosidase, glucagon,glucocerebrosidase, granulocyte macrophage colony stimulating factor(GM-CSF), phospholipase-activating protein (PLAP), gonadotropinchorionic (hCG), hemoglobins, hepatitis B vaccines, hirudin,hyaluronidases, idurnonidase, immune globulins, influenza vaccines,interleukins (1 alfa, 1beta, 2, 3, 4, 6, 10, 11, 12), IL-1 receptorantagonist (rhIL-1ra), insulins, interferons (alfa 2a, alfa 2b, alfa 2c,beta 1a, beta 1b, gamma 1a, gamma 1b), keratinocyte growth factor (KGF),transforming growth factors, lactase, leuprolide, levothyroxine,luteinizing hormone, lyme vaccine, natriuretic peptide, pancrelipase,papain, parathyroid hormone, PDGF, pepsin, platelet activating factoracetylhydrolase (PAF-AH), prolactin, protein C, octreotide, secretin,sermorelin, superoxide dismutase (SOD), somatropins (growth hormone),somatostatin, streptokinase, sucrase, tetanus toxin fragment, tilactase,thrombins, thymosin, thyroid stimulating hormone, thyrotropin, tumornecrosis factor (TNF), TNF receptor-IgG Fc, tissue plasminogen activator(tPA), TSH, urate oxidase, urokinase, vaccines, and plant proteins suchas lectins and ricins.

Also included herein is any synthetic polypeptide or any portion of apolypeptide with in vivo bioactivity. Furthermore, proteins prepared byrecombinant DNA methodologies including mutant versions ofaforementioned proteins, antibody fragments, single chain bindingproteins, catalytic antibodies and fusion proteins are included.

Linker

It is preferred for the linking agent to form a reversible linkage tothe biologically active moiety, preferably in such a fashion that aftercleavage of the linker, the biologically active moiety is released in anunmodified form. A variety of different linking agents or linking groupsmay be applied for this purpose; see B. Testa et al. (B. Testa, J.Mayer, Hydrolysis in Drug and Prodrug Metabolism, Wiley-VCH, 2003).

In a preferred embodiment, the linker is a cascade prodrug linkerconstituted of a masking group and an activating group. The biologicallyactive moiety is bound to the activating group, preferably through acarbamate bond. The release of the biologically active moiety iseffected by a two-step mechanism. In the first step, the masking groupis detached from the linker by cleavage of the bond connecting themasking group and activating group. The bond connecting the maskinggroup and the activating group may also be a carbamate bond.Subsequently, in a second step, the bond between biologically activemoiety and the activating group is cleaved, and the biologically activemoiety is released. As this second step is faster than the first step,the cleavage of the masking group is the rate-limiting step of therelease of the biologically active moiety.

The cleavage of the masking group is preferably based on a hydrolyticprocess, most preferably catalyzed by a nucleophile present in themasking group. In an autocatalytic fashion, this nucleophile attacks inan intramolecular fashion the carbon of the carbamate group constitutingthe linkage between the masking group and the activating group. Thepresence of the nucleophile in the vicinity of the carbamate groupenhances the susceptibility of the carbamate group to hydrolysis. In apreferred embodiment, the nucleophile is a tertiary amine which does notundergo a reaction with the carbamate carbonyl and does not lead to acyclization product.

Release of the biologically active moiety is initiated by anintramolecular rearrangement of the 1,6-elimination type, followed byautohydrolysis.

It is also preferred that at least part of the linker remains attachedto the hydrogel after cleavage of the bond with the biologically activemoiety. If the linker is a cascade prodrug linker, it is preferred forthe activating group to remain stably bound to the hydrogel.

Reactive Mesoporous Hydrogel

Hydrogels are three-dimensional, hydrophilic or amphiphilic polymericnetworks capable of taking up large quantities of water. The networksare composed of homopolymers or copolymers and are insoluble due to thepresence of covalent chemical or physical (ionic, hydrophobicinteractions, entanglements) crosslinks. The crosslinks provide thenetwork with structure and physical integrity.

Such reactive mesoporous hydrogels are characterized by the followingstructural components: crosslinking moiety, backbone moiety, reactivefunctional groups, pores, and optionally biodegradable bonds.

Backbone and Crosslinking Moieties

Non-limiting examples for suitable polymers for the synthesis ofhydrogels are chemically or physically crosslinked functionalized ornon-functionalized polyalkyloxy-based polymers like poly(propyleneglycol) or poly(ethylene glycol), dextran, chitosan, hyaluronic acid andderivatives, alginate, xylan, mannan, carrageenan, agarose, cellulose,starch, hydroxyethyl starch (HES) and other carbohydrate-based polymers,poly(vinyl alcohols), poly(oxazolines), poly(anhydrides), poly(orthoesters), poly(carbonates), poly(urethanes), poly(acrylic acids),poly(acrylamides) such as poly(hydroxypropylmethacrylamide) (HMPA),poly(acrylates), poly(methacrylates) likepoly(hydroxyethylmethacrylate), poly(organophosphazenes),poly(siloxanes), poly(vinylpyrrolidone), poly(cyanoacrylates),poly(esters) such as poly(lactic acid) or poly(glycolic acids),poly(iminocarbonates), poly(amino acids) such as poly(glutamic acid) orpoly lysine, collagen, gelatin, copolymers, grafted copolymers,cross-linked polymers, hydrogels, and block copolymers from the abovelisted polymers.

These polymers may serve as backbone moieties or crosslinking moieties.In addition to oligomeric or polymeric crosslinking moieties,low-molecular crosslinking moieties may be used, especially whenhydrophilic high-molecular weight backbone moieties are used for thehydrogel formation.

Suitable physical or chemical crosslinking methods are known to theperson skilled in the art and are described in W. E. Hennink and C. F.van Nostrum, Adv. Drug Del. Rev. 2002, 54, 13-36.

Pores

The chains of the network in a mesoporous hydrogel are connected in sucha fashion that pores exist in the hydrated state and that a substantialfraction of these pores are of dimensions between 1 and 100 nm.

The hydrogel is mesoporous with respect to the biologically activemoiety (e.g. drug molecule) to be carried, i.e. the average size of thepore of the hydrogel is larger than the diameter of the biologicallyactive moiety. For instance, a hydrogel that is mesoporous with respectto insulin molecules has pores of more than 4 nm in size (in thehydrated state).

The dimensions of the pores may be controlled by adjusting both lengthof crosslinker and degree of crosslinking.

For example, if small molecule crosslinkers are used for instance on abiopolymer such as dextran, porosity may be controlled through thedegree of crosslinking. Usually, the lower the degree of crosslinking isthe larger the size of the pores.

The size of the pore increases with crosslinker length. Crosslinkerlength refers to the spacer length between the two reactive groups usedfor the crosslinking of the backbone moiety. A typical polymericcrosslinker for the mesoporous hydrogel has at least two functionalgroups, usually at the ends of the polymeric chain. The functionalgroups are usually connected by a linear or branched chain of MW between500 and 50000. Such crosslinkers may be macromonomers, in which case themacromonomers are characterized by having at least two polymerizablefunctional groups.

Functional Groups

The hydrogel is a functionalized material. The reactive functionalgroups serve as conjugation sites for the linker Ideally, the reactivefunctional groups are dispersed homogeneously throughout the hydrogel,and may or may not be present on the surface of the hydrogel.

Non-limiting examples of such reactive functional groups include but arenot limited to carboxylic acid and activated derivatives, amino,maleimide, thiol, sulfonic acid and derivatives, carbonate andderivatives, carbamate and derivatives, hydroxyl, aldehyde, ketone,hydrazine, isocyanate, isothiocyanate, phosphoric acid and derivatives,phosphonic acid and derivatives, haloacetyl, alkyl halides, acryloyl andother alpha-beta unsaturated michael acceptors, arylating agents likearyl fluorides, hydroxylamine, disulfides like pyridyl disulfide, vinylsulfone, vinyl ketone, diazoalkanes, diazoacetyl compounds, epoxide,oxirane, and aziridine.

Preferred functional groups for the polymer include, but are not limitedto, thiol, maleimide, amino, carboxylic acid and derivatives, carbonateand derivatives, carbamate and derivatives, aldehyde, and haloacetyl.

In a preferred embodiment of the invention, the reactive mesoporoushydrogel is in the form of a shaped article such as a mesh or a stent.Most preferably, the hydrogel is formed into microparticulate beads thatcan be administered as subcutaneous or intramuscular injectably by meansof a standard syringe. Such soft beads may have a diameter of between 1and 500 micrometers.

Biodegradable Bonds

Biodegradability of the mesoporous hydrogel is of importance if thehydrogel is to be used for medical applications such as wound healing,wound sealing or for drug delivery (or, indeed, delivery of any type ofbiologically active moieties). In such applications, the hydrogel isadministered by a subcutaneous or intramuscular injection or appliedtopically to a wound and left in the organism to be degraded in vivo andresorbed or excreted.

For biodegradability of the hydrogel, biodegradable bonds have to beincorporated into the backbone and/or crosslinking moieties. Thesusceptibility of these biodegradable bonds to cleavage under in vivoconditions may cause complete degradation of the hydrogel after acertain time period, which is desirable for the abovementionedapplications in the medical field. Cleavage of these biodegradable bondsmay be enzymatically or chemically triggered, or be a combination ofboth.

Biodegradable bonds which may be cleaved chemically under in vivoconditions include, but are not limited to, phosphate, phosphonate,carbonate, carbamate, disulfide and ester bonds.

There exists a huge variety of bonds that may be cleaved enzymatically.Hydrogels with biopolymer backbones or biopolymer crosslinkers are perse biodegradable on the surface of the hydrogel article if enzymes arepresent for which the backbone chains are substrates. The rate ofdegradation under in vivo conditions is different for every differenttype of hydrogel. In general, the degradation rate is a function of thedegradability of the backbone (number of cleavable bonds, dependence ofbond cleavage on autohydrolysis or enzymatic catalysis) and the degreeof crosslinking. Even though crosslinks do not directly contribute tothe degradability of the hydrogel, the crosslinks can enable enzymeaccess into the hydrogel if the degree of crosslinking is small enoughthat the pores are large enough for the enzymes to penetrate into thehydrogel. Suitable biopolymers include, but are not limited to,carbohydrate-based polymers like dextran, chitosan, hyaluronic acid andderivatives, alginate, xylan, mannan, carrageenan, agarose, cellulose,starch, and hydroxyethyl starch and poly- or oligopeptide basedoligomers or polymers like synthetic peptide sequences, collagen andgelatin.

MHP Preparation Process

In order to guarantee that the prodrug of the biologically active moietyis only coupled to mesopores of the hydrogel, the biologically activemoiety has to be reacted with the hydrogel after the hydrogel has beensynthesized. This ensures that the release of the biologically activemoiety from the hydrogel is governed by the prodrug linker and isindependent from the optional hydrogel degradation. This is becauseafter cleavage of the prodrug linker the biologically active moiety canfreely diffuse out of the mesopores of the hydrogel.

A further advantage of this method of preparation is that reagents andsolvents contacted with the hydrogel during the preparation of thehydrogel may be removed from the hydrogel after completion of thepreparation by a filtration step. Efficient reagent and solvent removalavoid denaturation or modification of the biologically active moietyadded to the hydrogel. Efficient reagent and solvent removal also avoidsleakage of potentially toxic substances after administration to anorganism.

Representative examples for the preparation of MHP are given in theexamples section. MHPs can also be prepared by several other methods.

To prepare a MHP (Method A in FIG. 1), a prodrug linker agent can becoupled to the reactive mesoporous hydrogel in a first reaction step.Such a suitable prodrug linking agent carries two functional groups. Thefirst one of the functional groups would serve as the attachment of theprodrug linker to the hydrogel, and the second one of the functionalgroups would subsequently be conjugated to the biologically activemoiety through a suitable functional group present in the biologicallyactive moiety.

Such first reactive functional groups should be complementary to afunctional group present in the reactive mesoporous hydrogel.Non-limiting examples of such first reactive functional groups include,but are not limited to, carboxylic acid and activated derivatives,amino, maleimide, thiol, sulfonic acid and derivatives, carbonate andderivatives, carbamate and derivatives, hydroxyl, aldehyde, ketone,hydrazine, isocyanate, isothiocyanate, phosphoric acid and derivatives,phosphonic acid and derivatives, haloacetyl, alkyl halides, acryloyl andother alpha-beta unsaturated Michael acceptors, arylating agents likearyl fluorides, hydroxylamine, disulfides like pyridyl disulfide, vinylsulfone, vinyl ketone, diazoalkanes, diazoacetyl compounds, epoxide,oxirane, and aziridine.

Preferred first functional groups of the prodrug linker include thiol,maleimide, amino, carboxylic acid and derivatives, carbonate andderivatives, carbamate and derivatives, aldehyde, and haloacetyl.

After activation of the second one of the functional groups of theprodrug linker, the linker-hydrogel conjugate may be contacted with thebiologically active moiety in the second reaction step and excessbiologically active moiety (e.g., excess drug) may be removed byfiltration after conjugation of the biologically active moiety to thehydrogel-bound prodrug linker. Despite the large size of the pore of thehydrogel, the biologically active moiety remains bound inside thehydrogel by the covalent attachment of a suitable functional grouppresent on the biologically active moiety to the second functional groupof the prodrug linker.

Suitable second functional groups of the prodrug linker include, but arenot limited to, carboxylic acid and derivatives, carbonate andderivatives, hydroxyl, hydrazine, hydroxylamine, maleamic acid andderivatives, ketone, amino, aldehyde, thiol and disulfide.

Suitable functional groups present on the biologically active moietyinclude, but are not limited to, thiol, carboxylic acid, amino,hydroxyl, ketone, and imidazole.

Optionally this reaction sequence may be inverted, and the prodruglinker may be first conjugated to the biologically active moiety and theresulting biologically active moiety-prodrug linker conjugate may thenreact with the reactive mesoporous hydrogel (Method B in FIG. 1).

These preparation methods are shown schematically in FIG. 1.

Reactive Mesoporous Hydrogel Synthesis Process

Hydrogels which are reactive and mesoporous may be prepared by a varietyof different methods. One particular synthesis process is based on usinga crosslinking macromonomer carrying at least two polymerizablefunctional groups and a non-crosslinking macromonomer or monomercarrying one polymerizable functional group and at least one functionalgroup that is not intended to participate in the polymerization step.Additional diluent monomers may or may not be present. Copolymerizationof these components results in a hydrogel containing functional groupsprovided by the non-crosslinking macromonomer. In order to ensure thatthe functional group is available for reactions after completion of thepolymerization, the conditions for polymerization are chosen such thatthe functional group is not modified. Alternatively, the functionalgroup may be protected by use of a reversible protecting group known tothe person skilled in the art, which is removed after thepolymerization.

Useful polymerizable functional groups include, but are not limited to,radically polymerizable groups like vinyl, vinyl-benzene, acrylate,acrylamide, methacylate, methacrylamide and ionically polymerizablegroups like oxetane, aziridine, and oxirane.

In an alternative method of preparation, the hydrogel is generatedthrough chemical ligation reactions. The hydrogel may be formed from twomacromolecular educts with complementary functionalities which undergo areaction such as a condensation or addition. One of these startingmaterials is a crosslinker with at least two identical functional groupsand the other starting material is a homomultifunctional backbonestructure. Suitable functional groups present on the crosslinker includeterminal amino, carboxylic acid and derivatives, maleimide and otheralpha,beta unsaturated Michael acceptors like vinylsulfone, thiol, andhydroxyl groups. Suitable functional groups present in the backbonestructure include, but are not limited to, amino, carboxylic acid andderivatives, maleimide and other alpha,beta unsaturated Michaelacceptors like vinylsulfone, thiol, and hydroxyl groups.

If the crosslinker reactive functional groups are usedsubstoichiometrically with respect to backbone reactive functionalgroups, the resulting hydrogel will be a reactive hydrogel with freereactive functional groups attached to the backbone structure.

MHP with Staged Release and Degradation Kinetics

This invention also includes degradable mesoporous hydrogel prodrugsexhibiting minimal release of biologically active moiety conjugated tohydrogel degradation products.

In general, it is of advantage to limit the structural diversity ofdegradation products of a polymeric drug formulation with respect tochain lengths, substitutions or modifications. Specifically, the releaseof biologically active moiety-polymer conjugates should be avoided.

Hydrogel degradation may result in the release of degradation productconjugates of the biologically active moiety tethered to degradationproducts by means of the prodrug linker if degradation kinetics of thehydrogel are of a similar order as prodrug cleavage kinetics. Thedegradation product conjugates (shown schematically in FIG. 4) areundesired modifications of the biologically active moieties. Only few ofthese degradation product conjugates will appear if the cleavage ofdegradable hydrogel is at least one order of magnitude slower than therelease of the biologically active moiety. There may, however, beapplications, such as weekly injections of MHP in which a more rapiddisintegration of the hydrogel is desirable.

It has now been surprisingly found that through controlling the degreeof crosslinking, the degree of chain length of the backbone polymer, thepositioning of degradable bonds and the site of the prodrug linker, therelease kinetics of the resulting degradation products from the hydrogelcan be fine-tuned and the heterogeneity of degradation products can beminimized.

In such a hydrogel with controlled degradation properties, thedegradable bonds are exclusively located in the crosslinking chains. Ina most preferred embodiment, there are precisely two degradable bondsper linear crosslinking chain and they are positioned between the chainand backbone attachment site. The crosslinker carrying the degradablebonds is symmetrical with respect to the degradable bonds, rendering thebonds chemically identical. Such a biodegradable mesoporous hydrogelprodrug is shown schematically in FIG. 2.

Functional groups for attachment of the prodrug linker are positioned onside chains emanating from the backbone polymer. There are no degradablebonds between these functional groups and the non-biodegradable backboneor in the non-biodegradable backbone itself.

Cleavage of the degradable bonds of the crosslinker results in atwo-stage process. Shortly after initial time only cleavage productscontaining crosslinker units are released from the hydrogel when eachnon-biodegradable backbone is linked with at least one othernon-biodegradable backbone via several crosslinkers. If, for instance,esters of PEG have been used, the released compound is PEG. The releaseof PEG from this type of hydrogel follows approximately first orderkinetics.

After a certain lag time, backbone structures are released. Unlike therelease of crosslinker cleavage product, the release of the backbonestructures follows sigmoidal kinetics. The released backbone is a linearchain substituted with residues formerly connected to crosslinkingmoieties (for instance side chains terminating with carboxylic acids)and the functional group moiety. FIG. 3 shows schematically a partiallydegraded (FIG. 3 a) and a fully degraded (FIG. 3 b) MHP.

It is desirable to prolong the lag time to such an extent, that almostall drug release (>90%) has occurred before a significant amount ofrelease of the backbone (<10%) has taken place. This lag time can becontrolled by adjusting the number of crosslinks and the halflife of thebiodegradable bond. The lag time can be increased by incorporating morecrosslinks per backbone and increasing the half-life of thebiodegradable bond. The effect of an increased lag time by increasingthe number of crosslinks is shown in FIG. 10.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the preparation process for mesoporous hydrogel prodrugs

FIG. 2 shows schematically the structure of biodegradable mesoporoushydrogel prodrugs

FIG. 3 shows the degradation process of biodegradable mesoporoushydrogel prodrugs

FIG. 4 shows schematically an undesired polymer modified prodrug

FIG. 5 shows the insulin release from a polyacrylamide-based mesoporoushydrogel prodrug

FIG. 6 shows the insulin release from a carbohydrate-based mesoporoushydrogel prodrug

FIG. 7 shows the in vivo release of insulin from mesoporous hydrogelprodrugs

FIG. 8 shows the insulin release from a biodegradable mesoporoushydrogel prodrug

FIG. 9 shows the LCMS characterization of released insulin afterexplantation

FIG. 10 shows the degradation of biodegradable hydrogels

EXAMPLES Maleimide Derivatization of Polyacrylamide BasedNon-Biodegradable Reactive Mesoporous Hydrogel (Amino-PEGA)

Non-biodegradable mesoporous NH₂-PEGA hydrogel beads with 0.4 mmol/gloading and 150-300 μm bead size were purchased from Novabiochem.NH₂-PEGA Versamatrix-800 hydrogel beads with 0.31 mmol/g loading and80-100 μm bead size were obtained from Versamatrix (Denmark).

2.5 g methanol-wet NH₂-PEGA-hydrogel (0.4 mmol/g NH₂-loading) wasweighed into a syringe equipped with a polypropylene frit. Maleimideloading was adjusted by acylation employing a mixture of activatedmaleimidopropionic acid and acetic acid as described in the following.The hydrogel was washed 5 times with DMF and reacted with 13.5 mg (0.08mmol) 3-maleimidopropionic acid, 115.2 μl (1.92 mmol) acetic acid and313 μl (2 mmol) DIC in 4 ml DMF for 30 min. The maleimide derivatizedhydrogel 1a was washed 10 times with DMF and DCM and finally withacetonitrile.

Hydrogel 1b was synthesized following the protocol above with thefollowing modification. 2.5 g methanol-wet NH₂-PEGA-hydrogel (˜250 mgdry resin) was reacted with 6.8 mg (0.04 mmol) 3-maleimidopropionicacid, 117.6 μl (1.96 mmol) acetic acid and 313 μl (2 mmol) DIC in 4 mlDMF for 30 min. Finally the hydrogel was washed as described.

Hydrogel 1c was synthesized following the protocol above with thefollowing modification. 2 g methanol-wet NH₂-PEGA Versamatrix-800hydrogel (0.31 mmol/g NH₂-loading, dry resin) was washed as describedand reacted with 25.3 mg (0.15 mmol) 3-maleimidopropionic acid, 115.2 μl(1.85 mmol) acetic acid and 313 μl (2 mmol) DIC in 4 ml DMF for 30 min.Finally the hydrogel was washed as described.

Synthesis of rh-Insulin Loaded PEGA Hydrogel 4 and 5a,b

2 and 3 were synthesized as described in co-pending UK patentapplication No. 0415043.9 30 mg of maleimide derivatized resin 1b(loading 15 μmol/g, 450 nmol) was reacted with 3 mg of compound 2 (480nmol, 1.06 eq) in 600 μl 20/80 (v/v) acetonitrile/50 mM phosphate buffer(pH 7.4) for 10 min to give rh-insulin loaded hydrogel 4. The hydrogel 4was washed 5 times with 50/50 (v/v) acetonitrile/water and three timeswith acetonitrile and dried under vacuum.

Synthesis of hydrogel 5a or 5b by reacting 1 equivalent compound 3 inrelation to the theoretical amount of maleimide groups on the hydrogelwith hydrogel 1b or 1c, respectively, followed the synthesis protocolabove.

In Vitro rh-Insulin Release Experiments from Non-BiodegradableMesoporous Hydrogel Prodrug 4

4 mg of 4 was weighed into a test tube and incubated with 1000 μl 10 mMHEPES buffer pH 7.4, 150 mM NaCl, 0.005% Tween at 37° C. 45 μl sampleswere taken at regular intervals and quantitatively analyzed forrh-insulin by a RP-HPLC assay. The rh-insulin peak was integrated andrh-insulin concentration was obtained from a standard curve. A firstorder release kinetic was fitted to the data points to give the linkerhalf life and maximal rh-insulin release at t=∞ (FIG. 5).

Release of rh-insulin from PEGA hydrogel 5a or 5b was carried out asdescribed above.

In Vivo Release Experiments of rh-Insulin from Non-BiodegradableMesoporous Hydrogel Prodrugs 4 and 5a

In vivo studies were conducted at the “Steinbeis-Transfer-ZentrumBiopharmazie and Analytik—Heidelberg” in male and female Wistar rats.

The rats weighing 200-300 g were kept under standard conditions and fedad libitum. Blood samples (1500) were collected from the retro-orbitalplexus and a suspension of rh-insulin loaded hydrogel in 300-4000 PBSwas subcutaneously administered in the upper hind leg area. Blood wasdrawn at different times after administration. Animals were lightlyanesthetized by inhaled isoflurane during all blood draws andinjections. All blood samples were collected into tubes containing EDTAand centrifuged. Plasma was separated and stored at −18° C. untilassayed. rh-Insulin concentration was determined from plasma samplesusing the species specific sandwich human insulin ELISA kit (Mercodia,Sweden). The results were statistically analyzed and plasma rh-insulinconcentrations were plotted over time after administration (FIG. 7).

Total rh-insulin dose/ Sample Hydrogel rat (calculated from Hydrogelvolume dose/rat in vitro release) N rats 5a 400 μl   9 mg ~26 nmol 3 4 300 μl 10.7 mg ~38 nmol 2Explantation of Administered Mesoporous Hydrogel Prodrug 5b andInvestigation of rh-Insulin Integrity

Hydrogel administration, blood sampling and determination of therh-insulin concentration followed the protocol described above.

Total rh-insulin dose/ Hydrogel Sample Hydrogel rat (calculated fromsample volume dose in vitro release) N rats 5b 400 μl 16 mg ~138 nmol 1

After 6 days the rat was sacrificed. The hydrogel was explanted andwashed 10 times with water, 10 times with 50/50 (v/v) acetonitrile/waterand dried under vacuum. About 1 mg of hydrogel was weighed into a testtube and incubated with 400 μl 100 mM Tris/HCl buffer pH 9, at 37° C.After 24 h the solution was separated from the hydrogel and analyzed forrh-insulin and enzymatic degradation products thereof by LC/MS (FIG. 9).The strong peak at 4.2 min elution time can be assigned to rh-insulinaccording to the mass spectrum. Smaller peaks before and after therh-insulin peak correspond to the A- and B-chain of rh-insulin,respectively. No rh-insulin related degradation products were detected.A- and B-chain were also detected in in vitro release experiments at pH9 and thus do not indicate enzymatic degradation.

Synthesis of Biodegradable Reactive Mesoporous Hydrogel 8 Synthesis ofMacromonomer bis-acryl-glycyl-PEG900 (6)

10 g PEG900 (11.1 mmol) were dissolved in 50 ml DCM and the solution wascooled to 0° C. A solution of 3.9 g (23.3 mmol) boc-glycine, 5.67 g(46.6 mmol) DMAP, and 950 mg DCC (4.6 mmol) in 50 ml DCM was added andthe mixture was stirred over night at room temperature. The solidbyproduct dicyclohexyl urea was filtered off and the filtrate was washedtwo times with 1 M HCl and two times with water. The organic phase wasdried over sodium sulfate and was concentrated in vacuo to approximately50 ml. 50 ml TFA were added at 0° C. and the solution was stirred atroom temperature for 30 min to remove the boc protecting groups. Thesolvent was removed under reduced pressure and the resulting oil wasredissolved in 50 ml DCM. 5 ml 2 M HCl in diethyl ether was added andthe product precipitated by the addition of 400 ml diethyl ether andcollected by centrifugation. The resulting oil was dissolved in 100 ml0.1 M aqueous HCl and lyophilized (yield: 9.5 g) LC/MS: [M+H]⁺=882.3,926.4, 970.4, 1014.4, 1058.4, 1102.5, 1146.6 (MW+Hcalculated=1014.2+/−x*44.05 g/mol)

1.4 g bis-glycyl-PEG900 were dissolved in 15 ml DCM and 750 μl (5.32mmol) triethylamin and 220 μl (2.66 mmol) acryl chloride were added at0° C. The mixture was stirred at room temperature for 30 min and 35 mlDCM were added. The organic phase was washed with 1 M aqueous HCl and 5times with water. The organic phase was dried over sodium sulfate andconcentrated under reduced pressure to approximately 10 ml. The product6 was precipitated by the addition of 60 ml 1/1 (v/v) diethylether/heptane and collected by centrifucation (yield 1.05 g).

LC/MS: [M+Na]⁺=1012.0, 1056.6, 1100.5, 1144.5, 1188.6, 1232.5, 1275.6,1319.7

(MW+Na calculated=1144.2+/−x*44.05 g/mol)

Synthesis of N-boc,N′-acryloyl-4,7,10-trioxamidecane-1,13-diamine

2 g (9.1 mmol) 4,7,10-trioxamidecane-1,13-diamine were dissolved in 15ml DCM and 1 g (4.6 mmol) di-tert.-butyl-dicarbonate in 10 ml DCM wasadded dropwise at 0° C. The solution was stirred for 2 h at roomtemperature and the organic phase was washed five times with water. Theorganic phase was dried over sodium sulfate and concentrated underreduced pressure to approximately 10 ml.Mono-boc-4,7,10-trioxamidecane-1,13-diamine was precipitated ashydrochloride salt by the addition of 2 M HCl in diethyl ether (yield:1.1 g, 3.1 mmol, 67%).

1.1 g (3.1 mmol) mono-boc-4,7,10-trioxamidecane-1,13-diamine wasdissolved in 10 ml DCM and 900 μl (6.2 mmol) triethylamine was added.The mixture was cooled to 0° C. and 260 μl (3.2 mmol) acryl chloride in10 ml DCM was added dropwise. The solution was stirred at roomtemperature for 30 min and the organic phase was washed two times with0.1 M aqueous HCl. The organic phase was dried over sodium sulfate andconcentrated to 7 ml under reduced pressure. The product 7 wasprecipitated by the addition of 50 ml 1/1 (v/v) diethyl ether/heptaneand collected by centrifugation. (yield: 940 mg, 2.5 mmol, 78%).

LC/MS: [M+Na]⁺=398.1 (MW+Na calculated=397.4 g/mol)

Biodegradable Reactive Mesoporous Hydrogel Formation

100 mg (100 μmol) compound 6, 6 mg (15 μmol) compound 7 and 21 mg (212μmol) N,N-dimethylacrylamide were dissolved in 500 μl 50 mM phosphatebuffer (pH 7.0) in a test tube. After addition of 30 μl 1 M ammoniumperoxodisulfate (APS), the solution was vortexed and polymerization wasinitiated by addition of 80 μl 2 M N,N,N′,N′-tetramethylethylenediamine(TEMED)/HCl, pH 7.0. The spontaneously formed hydrogel was incubated forfurther 30 min, ground to particles <1 mm and transferred into a syringeequipped with a polypropylene frit. After extensive washing of thehydrogel with water, DMF and DCM (5 times each), boc-protecting groupswere cleaved by incubation with (v/v) 50/50 TFA/DCM for 10 min. Finally,the hydrogel 8 was washed five times with DCM, five times with DMF, oncewith 1/99 (v/v) DIEA/DMF and five times with DMF.

Synthesis of Maleimide Derivatized Biodegradable Polyacrylamide BasedMesoporous Hydrogel 9

100 mg ground biodegradable hydrogel 8 was weighed into a syringeequipped with a polypropylene frit. Maleimide derivatization wasachieved by acylation with 101 mg (0.6 mmol) maleimidopropionic acid, 94μl (0.6 mmol) DIC in 2 ml DMF for 30 min to give maleimide derivatizedhydrogel 9. The hydrogel was washed 10 times with DMF and DCM.

Synthesis of Fluorescein-Carboxamido-Lys(B29)-Rh-Insulin LoadedBiodegradable Polyacrylamide Based Mesoporous Hydrogel Prodrug 11

Compound 10 was synthesized as described in co-pending UK patentapplication No. 0415043.9, the disclosure of which is incorporated byreference. 0.25 mg of compound 10 (35 nmol) was dissolved in 100 μl40/40/20 (v/v/v) acetonitrile/water/0.5 M phosphate buffer pH 7.0. Thesolution was incubated for 3 min with 5.6 mg of maleimide derivatizedbiodegradable hydrogel 9 to givefluorescein-carboxamido-Lys(B29)-rh-insulin loaded hydrogel 11. Thehydrogel 11 was washed five times with 50/50 (v/v) acetonitrile/water,three times with acetonitrile and dried under vacuum.

In Vitro Fluorescein-Carboxamido-Lys(B29)-rh-Insulin Release Experimentsfrom Biodegradable Polyacrylamide Based Mesoporous Hydrogel Prodrug 11

The fluorescein-carboxamido-Lys(B29)-rh-insulin loaded biodegradablehydrogel 11 was incubated in 100 μl 10 mM HEPES buffer (pH 7.4), 150 mMNaCl, 0.005% Tween at 37° C. 80 μl samples were taken at regularintervals and quantitatively analyzed forfluorescein-carboxamido-Lys(B29)-rh-insulin by a RP-HPLC assay. A firstorder release kinetic was fitted to the data points to give the halflife of the prodrug linker and maximalfluorescein-carboxamido-Lys(B29)-rh-insulin concentration at t=∞ (FIG.8). The integrity of the releasedfluorescein-carboxamido-Lys(B29)-rh-insulin 12 was confirmed by RP-LCMSand SEC (data not shown).

Synthesis of Fluorescein Labeled Compound 12

0.3 g Sieber amide resin (loading 0.5 mmol/g) was weighed into a syringeequipped with a polypropylene frit. Fmoc-removal was achieved byincubation in 2/2/96 (v/v/v) piperidin/DBU/DMF for 10 min and the resinwas washed 5 times with DMF.

The resin was incubated for 1 h with 264 mg (0.45 mmol)Fmoc-Cys(Trt)-OH, 144 mg (0.45 mmol) TBTU and 157 μl DIEA (0.9 mmol) in3 ml DMF and washed 5 times with DMF. After fmoc-removal, Fmoc-Ado-OHwas coupled by incubation of 156 mg (0.45 mmol) Fmoc-Ado-OH 173 mg (0.45mmol) TBTU and 157 μl DIEA (0.9 mmol) in 3 ml DMF according to theprocedure above. Fmoc was removed and the resin was reacted with 338 mg(0.9 mmol) 5,6-carboxyfluorescein (isomeric mixture), 140 mg (0.9 mmol)HOBt and 141 μl (0.9 mmol) DIC in 3 ml DMF for 2 h. Finally the resinwas incubated in 2/2/96 (v/v/v) piperidine/DBU/DMF for 10 min, washed 10times in DCM and dried under vacuum.

12 was cleaved from the resin with 50/5/45 (v/v/v) TFA/TES/DCM for 30min and purified by RP-HPLC.

MS: [MH]⁺=625 g/mol (MW calculated=624 g/mol)

Degradation of Biodegradable Polyacrylamide-Based Mesoporous Hydrogel 13In Vitro

A 100 μM solution of compound 12 (35 nmol) in 500 μl 40/40/20 (v/v/v)acetonitrile/water/0.5 M phosphate buffer pH 7.0 was reacted with 5 mgof maleimide derivatized biodegradable hydrogel 9 for 5 min to give thefluorescein labeled hydrogel 13. The hydrogel 13 was washed 5 times with50/50 (v/v) acetonitrile/water and three times with acetonitrile anddried under vaccuum.

Hydrogel degradation experiment was performed at pH 9 to reduce the timeof degradation. Degradation time at physiological pH 7.4 was estimatedby a scaling factor 40 that accounts for the approx. 40 fold increasedhydroxide ion concentration at pH 9 compared to pH 7.4.

The hydrogel was suspended in 1 ml 50 mM borate buffer (pH 9.0), 150 mMNaCl, 0.005% Tween and incubated at 37° C. 60 μl samples were taken atdifferent time intervals and quantitatively analyzed for polymerbackbone coupled fluorescein 14 by photometry at 500 nm. The data show adelayed and sigmoidal release of 14 (data not shown).

Synthesis of rh-Insulin Loaded Carbohydrate-Based Mesoporous HydrogelProdrug 16 and In Vitro Release

NHS-activated “Sepharose 4 Fast Flow” hydrogel beads (chemicallycrosslinked agarose, crosslinker epichlorhydrin) were purchased fromAmersham.

1.5 g ethanol-wet Sepharose hydrogel (150 mg dry hydrogel) was weighedinto a syringe equipped with a polypropylene frit and reacted with 1 M4,7,10-trioxamidecan-1,13-diamin in DMF for 30 min. After 5 washingsteps with DMF, hydrogel was reacted with 8.5 mg (0.05 mmol)3-maleimidopropionic acid, 57 μl (0.95 mmol) acetic acid, 151 mg (1mmol) HOBt and 158 μl (1 mmol) DIC in 4 ml DMF for 30 min to givemaleimide derivatized hydrogel 15. The hydrogel 15 was washed 10 timeswith DMF and finally with acetonitrile.

1.5 mg 3 was dissolved in 25/75 (v/v) acetonitrile/50 mM phosphatebuffer pH 7.4 and reacted with 10.8 mg maleimide derivatized hydrogel 15for 10 min. The rh-insulin loaded hydrogel 16 was washed five times with50/50 (v/v) acetonitrile/water and three times with acetonitrile anddried under vacuum.

2 mg rh-insulin-loaded hydrogel 16 was suspended in 1000 μl 10 mM HEPESbuffer (pH 7.4), 150 mM NaCl, 0.005% Tween and incubated at 37° C. 60 μlsamples were taken at regular intervals and quantitatively analyzed forrh-insulin by an RP-HPLC assay. A first order release kinetic was fittedto the data points to give the half life of the prodrug linker andmaximal rh-insulin release at t=∞ (FIG. 6). The integrity of thereleased rh-insulin was confirmed by LCMS (data not shown).

Synthesis of Biodegradable Mesoporous Hydrogel 20 and 21 Synthesis ofBis-Mercaptoacetyl-Glycyl-PEG900 (17)

1 g (0.92 mmol) bis-glycyl-PEG900×2HCl was dissolved in 7 ml DMF and 600mg (2 mmol) SAMA-OPfp ester was added. The mixture was stirred at roomtemperature for 60 min. 200 μl hydrazin hydrate was added and themixture stirred for 5 min at room temperature to remove the acetylgroup. After addition of 400 μl acetic acid the product 17 was purifiedby RP-HPLC and lyophilized (yield 740 mg).

LC/MS:

[M+H]⁺=1030.2, 1074.2; 1118.3; 1162.2; 1206.3; 1250.3; 1294.4, 1338.4

(MW+H calculated=1161.4+/−x*44.05 g/mol)

Synthesis of 1-naphtylacetyl-tetra-Lys(Ado-mp)-amide (18) and1-naphtylacetyl-octa-Lys(Ado-mp)-amide (19)

18 was synthesized by standard solid-phase organic synthesis usingTBTU/DIEA activation as described for compound 12.

To 1 g TentaGel Sieber amide resin (0.17 mmol/g loading) four timesFmoc-Lys(ivDde)-OH and 1-naphthyl acetic acid was coupled by TBTU/DIEAactivation using 3 equivalents amino acid in relation to free aminogroups on the resin. To remove the ivDde protecting group the resin wasincubated three times with 4% hydrazine in DMF for 7 min. After washingof the resin with DMF, Fmoc-Ado-OH was coupled to the amino groups byTBTU/DIEA activation. 3-maleimidopropionic acid was coupled by DICactivation using 3 equivalents 3-maleimidopropionic acid in relation tofree amino groups on the resin. Compound 18 was cleaved from the resinby incubation with 94/3/3 (v/v/v) DCM/triethylsilane/TFA for 60 min.After evaporation of the solvent, 18 was purified by RP-HPLC andlyophilized.

19 was synthesized as described above by coupling eightFmoc-Lys(ivDde)-OH residues instead of four residues.

LC/MS:

n=4:

[M+H]⁺=1883.9; [M+Na]⁺=1905.2 (MW calculated=1879.1 g/mol)

n=8:

[M+2Na]²⁺=1812; [M+H+Na]²⁺=1800; [M+2H]²⁺=1790 (MW calculated=3573.0g/mol)

Biodegradable Hydrogel 20 and 21 Formation

23.2 mg (20 μmol) 17 and 18.8 mg (10 μmol) 18 were dissolved in 150 μlwater and 50 μl 0.5 M sodium acetate buffer pH 5.5 were added. Thesolution was incubated at room temperature for 60 min.

The resulting hydrogel 20 was ground to particles <1 mm and transferredinto a syringe equipped with a polypropylene frit. The hydrogelparticles were washed five times each with 1/1 acetonitrile/water,water, and methanol and then dried under vacuum.

Hydrogel 21 was synthesized as described above using 23.3 mg (20 μmol)17 and 17.9 mg (5 μmol) 19.

Degradation Study

Hydrogel degradation experiments were performed at pH 9 to reduce thetime of degradation. Degradation time at physiological pH 7.4 wasestimated by a scaling factor 40 that accounts for the ˜40 foldincreased hydroxide ion concentration at pH 9 compared to pH 7.4.

50 mg of hydrogel 20 or 21 was suspended in 1 ml 50 mM sodium boratebuffer (pH 9.0), 150 mM NaCl, 0.005% Tween and incubated at 37° C. 30 μlsamples were taken at regular intervals and quantitatively analyzed forpolymer backbone coupled naphthyl 22 or 23 by photometry at 280 nm (FIG.10). The degradation kinetic shows a sigmoidal curve.

ABBREVIATION

-   Ado 8-amino-3,6-dioxa-octanoyl-   Boc t-butyloxycarbonyl-   DBU 1,3-diazabicyclo[5.4.0]undecene-   DCM dichloromethane-   (iv) Dde 1-(4,4-dimethyl-2,6-dioxo-cyclohexyliden)-3-methyl-butyl-   DIC diisopropylcarbodiimide-   DIEA diisopropylethylamine-   DMF N,N-dimethylformamide-   EDTA ethylenediaminetetraacetic acid-   fmoc 9-fluorenylmethoxycarbonyl-   Fmoc-Ado-OH Fmoc-8-amino-3,6-dioxaoctanoic acid-   HEPES N-(2-hydroxyethyl)piperazine-N-(2-ethanesulfonic acid)-   HOBt N-hydroxybenzotriazole-   LCMS mass spectrometry-coupled liquid chromatography-   mp 3-maleimidopropionyl-   MS mass spectrum-   MW molecular mass-   PEG poly(ethylene glycol)-   RP-HPLC reversed-phase high performance liquid chromatography-   RT room temperature-   SAMA-OPfp S-acetyl-mercaptoacetic acid pentafluorophenyl ester-   SEC size exclusion chromatography-   TBTU O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium    tetrafluoroborate-   TES triethylsilane-   TFA trifluoroacetic acid

The various publications, patents, patent applications and publishedapplications mentioned in this application are hereby incorporated byreference herein.

While there have been described what are presently believed to be thepreferred embodiments of the invention, those skilled in the art willrealize that changes and modifications may be made without departingfrom the spirit of the invention. It is intended to claim all suchchanges and modifications as fall within the true scope of theinvention.

What is claimed is:
 1. A mesoporous hydrogel-biologically active moietyconjugate prepared by a process comprising the steps of: synthesizingthe mesoporous hydrogel, wherein the mesoporous hydrogel comprisesbackbone and crosslinking moieties, wherein each backbone moiety islinked with at least one other backbone moiety via several crosslinkingmoieties and wherein the crosslinking moieties comprise biodegradablebonds; and after completing all said synthesizing covalently connectinga prodrug linker to the mesoporous hydrogel which prodrug linker forms areversible linkage to the biologically active moiety; and conjugating abiologically active moiety to the prodrug linker; wherein the connectingand the conjugating can be carried out in either order.
 2. Themesoporous hydrogel-biologically active moiety conjugate of claim 1,wherein the prodrug linker has two functional groups, a first one of thetwo functional groups being complementary to a functional group attachedto the mesoporous hydrogel and a second one of the two functional groupsbeing conjugable to the biologically active moiety.
 3. The mesoporoushydrogel-biologically active moiety conjugate of claim 2, wherein thefirst one of the two functional groups is selected from the group offunctional groups consisting of carboxylic acid, amino, maleimide,thiol, sulfonic acid, carbonate, carbamate, hydroxyl, aldehyde, ketone,hydrazine, isocyanate, isothiocyanate, phosphoric acid, phosphonic acid,haloacetyl, alkyl halides, acryloyl, alpha-beta unsaturated michaelacceptors, arylating agents, aryl fluorides, hydroxylamine, disulfides,vinyl sulfone, vinyl ketone, diazoalkanes, diazoacetyl compounds,epoxide, oxirane, and aziridine.
 4. The mesoporous hydrogel-biologicallyactive moiety conjugate of claim 2, wherein the first one of the twofunctional groups is selected from the group of functional groupsconsisting of thiol, maleimide, amino, carboxylic acid, carbonate,carbamate, aldehyde, and haloacetyl.
 5. The mesoporoushydrogel-biologically active moiety conjugate of claim 2, wherein thefirst one of the two functional groups comprises a thiol or maleimidegroup.
 6. The mesoporous hydrogel-biologically active moiety conjugateof claim 2, wherein the second one of the two functional groups isselected from the group of functional groups consisting of carboxylicacid, carbonate, hydroxyl, hydrazine, hydroxylamine, maleamic, ketone,amino, aldehyde, thiol and disulfide groups.
 7. The mesoporoushydrogel-biologically active moiety conjugate of claim 2, wherein thebiologically active moiety has a moiety functional group complimentaryto the second one of the two functional groups.
 8. The mesoporoushydrogel-biologically active moiety conjugate of claim 7, wherein themoiety functional group is selected from the group of functional groupsconsisting of thiol, carboxylic acid, amino, hydroxyl, ketone andimidazole.
 9. The mesoporous hydrogel-biologically active moietyconjugate of claim 1, wherein the biologically active moiety comprises abiopolymer.
 10. The mesoporous hydrogel-biologically active moietyconjugate of claim 1, wherein the biologically active moiety is selectedfrom the group of proteins or polypeptides consisting of ACTH, adenosinedeaminase, agalsidase, albumin, alpha-1 antitrypsin (AAT), alpha-1proteinase inhibitor (API), alteplase, anistreplase, ancrod serineprotease, antibodies (monoclonal or polyclonal, and fragments orfusions), antithrombin III, antitrypsins, aprotinin, asparaginases,biphalin, bone-morphogenic proteins, calcitonin (salmon), collagenase,DNase, endorphins, enfuvirtide, enkephalins, erythropoietins, factorVIla, factor III, factor VIlla, factor IX, fibrinolysin, fusionproteins, follicle-stimulating hormones, granulocyte colony stimulatingfactor (G-CSF), galactosidase, glucagon, glucocerebrosidase, granulocytemacrophage colony stimulating factor (GMCSF), phospholipase-activatingprotein (PLAP), gonadotropin chorionic (hCG), hemoglobins, hepatitis Bvaccines, hirudin, hyaluronidases, idurnonidase, immune globulins,influenza vaccines, interleukins (1 alpha, 1 beta, 2, 3, 4, 6, 10, 11,12), IL-1 receptor antagonist (rhIL-1ra), insulins, interferons (alpha2a, alpha 2b, alpha 2c, beta 1a, beta 1b, gamma 1a, gamma 1b),keratinocyte growth factor (KGF), transforming growth factors, lactase,leuprolide, levothyroxine, luteinizing hormone, lyme vaccine,natriuretic peptide, pancrelipase, papain, parathyroid hormone, PDGF,pepsin, platelet activating factor acetylhydrolase (PAF-AH), prolactin,protein C, octreotide, secretin, sermorelin, superoxide dismutase (SOD),somatropins (growth hormone), somatostatin, streptokinase, sucrase,tetanus toxin fragment, tilactase, thrombins, thymosin, thyroidstimulating hormone, thyrotropin, tumor necrosis factor (TNF), TNFreceptor-IgG Fc, tissue plasminogen activator (tPA), TSH, urate oxidase,urokinase, vaccines, and plant proteins.
 11. The mesoporoushydrogel-biologically active moiety conjugate of claim 1, wherein thebiologically active moiety comprises insulin.
 12. The mesoporoushydrogel-biologically active moiety conjugate of claim 1, wherein thebiologically active moiety comprises an organic small molecule bioactiveagent.
 13. The mesoporous hydrogel-biologically active moiety conjugateof claim 1, wherein the biologically active moiety is selected from thegroup of moieties consisting of central nervous system-active agents,anti-infective agents, anti-neoplastic agents, antibacterial agents,antifungal agents, analgesic agents, contraceptive agents,anti-inflammatory agents, steroidal agents, vasodilating agents,vasoconstricting agents, and cardiovascular agents with at least oneprimary or secondary amino group.
 14. The mesoporoushydrogel-biologically active moiety conjugate of claim 1, wherein thebiologically active moiety comprises an anti-sense or interferingnucleic acid.
 15. The mesoporous hydrogel-biologically active moietyconjugate of claim 1, wherein the hydrogel is synthesized from the groupof polymers consisting of polyalkyloxy-based polymers, dextran,chitosan, hyaluronic acid and derivatives, alginate, xylan, mannan,carrageenan, agarose, cellulose, starch, hydroxyethyl starch (HES) andother carbohydrate-based polymers, poly(vinyl alcohols),poly(oxazolines), poly(anhydrides), poly(ortho esters),poly(carbonates), poly(urethanes), poly(acrylic acids),poly(acrylamides), poly(acrylates), poly(methacrylates),poly(organophosphazenes), poly(siloxanes), poly(vinylpyrrolidone),poly(cyanoacrylates), poly(esters), poly(lactic acid), poly(glycolicacids), poly(iminocarbonates), poly(amino acids), collagen, and gelatin.16. The mesoporous hydrogel-biologically active moiety conjugate ofclaim 1, wherein the hydrogel comprises polyacrylamide or a derivativethereof.
 17. The mesoporous hydrogel-biologically active moietyconjugate of claim 1, wherein the hydrogel comprises poly(ethyleneglycol acrylamide) or a derivative thereof.
 18. The mesoporoushydrogel-biologically active moiety conjugate of claim 1, wherein thehydrogel is functionalized with a functional group selected from thegroup of reactive functional groups consisting of carboxylic acid,amino, maleimide, thiol, sulfonic acid, carbonate, carbamate, hydroxyl,aldehyde, ketone, hydrazine, isocyanate, isothiocyanate, phosphoricacid, phosphonic acid, haloacetyl, alkyl halides, acryloyl and otheralpha-beta unsaturated michael acceptors, arylating agents like arylfluorides, hydroxylamine, disulfides like pyridyl disulfide, vinylsulfone, vinyl ketone, diazoalkanes, diazoacetyl compounds, epoxide,oxirane, and aziridine.
 19. The mesoporous hydrogel-biologically activemoiety conjugate of claim 1, wherein the hydrogel is functionalized witha functional group selected from the group of functional groupsconsisting of thiol, maleimide, amino, carboxylic acid, carbonate,carbamate, aldehyde, and haloacetyl.
 20. The mesoporoushydrogel-biologically active moiety conjugate of claim 1, wherein thehydrogel is functionalized with maleimide.
 21. The mesoporoushydrogel-biologically active moiety conjugate of claim 1, wherein theprodrug linker is attached to a non-degradable backbone of themesoporous hydrogel.
 22. The mesoporous hydrogel-biologically activemoiety conjugate of claim 1 wherein the biodegradable bonds are selectedfrom the group of chemically-cleavable bonds consisting of phosphate,phosphonate, carbonate, carbamate, disulfide and ester bonds.
 23. Themesoporous hydrogel-biologically active moiety conjugate of claim 1,wherein the biodegradable bonds are enzymatically cleavable.